The present invention relates to a D-aminoacylase produced by fungi, a method for producing said D-aminoacylase, and a method for producing D-amino acids using said D-aminoacylase.
Enzymes have excellent catalytic functions with substrate specificity, reaction specificity, and stereospecificity. Stereospecificity of enzymes, with some exceptions, are nearly absolute.
Recent precise research has increased the importance of optically active substances for use in drugs, pesticides, feeds, and perfumes. Optical isomers sometimes have quite different biological activities. D(R)-form thalidomide has no teratogenic activity, but its L(S)-form shows strong teratogenicity. The practical use of thalidomide racemate caused the drug injury incidents. If one enantiomer shows an effective biological activity, the other enantiomer may sometimes have no activity, rather, it may competitively inhibit the activity of the effective enantiomer. In some cases, the activity of the racemate is reduced to half or less of the activity of the effective enantiomer. Accordingly, it is industrially important to obtain (synthesize or optically resolve) optically pure enantiomers. For this purpose, techniques for effective resolution of synthetic racemate have been widely used. Enzymatic optical resolution has drawn attention because it does not produce by-products and a bulk of liquid waste.
Generally, L-amino acids are widely and largely utilized in seasonings, food and feed additives, and infusions and are thus very highly demanded. L-Amino acids have been produced mainly by direct fermentation using microorganisms. Optical resolution is also a known method for producing L-amino acids by hydrolyzing N-acyl-DL-amino acids using L-aminoacylases. It has been utilized to industrially produce L-amino acids that are difficult to produce by fermentation. L-aminoacylases are widely found in animals, plants, and microorganisms. They have been purified from various organisms, and, their properties have been clarified. N-terminal amino acids of many proteins are considered to be N-acetylated in vivo. L-Aminoacylases presumably regenerate N-acetyl-amino acids produced by decomposition of proteins to amino acids. Among L-aminoacylases, acylase, which acts on N-acyl-L-glutamic acid, is reportedly involved in arginine biosynthesis (Fruth, H., Leisinger, T.: J. Gen. Microb. 125, pp1 (1981)).
In contrast, D-amino acids have not been a subject of interest for a long time because they are nonprotein amino acids. D-amino acids were known to occur in small cyclic peptides, peptidoglycan of bacterial cell walls, and peptide antibiotics. However, D-amino acids have been demonstrated to be constituents of neuro peptides and to exist as binding forms in tooth enamel, the lens, and cerebral proteins, resulting in investigation of physiological significance and enzymatic synthesis of D-amino acids.
At present, DL-amino acids have been optically resolved by physicochemical, chemical, and enzymatic methods. The enzymatic methods are the most convenient and industrially applicable for, for example, continuously producing L-methionine from N-acetyl-DL-methionine using a bioreactor of immobilized L-aminoacylase. D-amino acids may also be produced using hydantoinase. The method consists of two-step enzymatic reactions. The first reaction uses D-specific hydantoinase to convert D,L-5-substituted-hydantoin, which is synthesized at low cost from aldehyde analogues, to a D-carbamyl derivative. The second reaction uses D-amino acid carbamylase.
Another method comprises hydrolyzing N-acetyl-DL-amino acids with D-aminoacylase to produce D-amino acids (Sugie, M. and Suzuki, H.: Argric. Biol. Chem. 44, pp1089 (1980), Tsai, Y. C., Lin, C. S., Tseng, T. H., Lee, H. and Wang, Y. J.: J. Enzyme Microb. Technol. 14, pp384 (1992)).
D-aminoacylase was first reported to be found in Pseudomonas sp. KT83 isolated from soil by Kameda et. al in 1952 (Kameda, Y., Toyoura, H., Kimura, Y. and Yasuda, Y.: Nature 170, pp888 (1952)). This enzyme hydrolyzed N-benzoyl derivatives of D-phenylalanine, D-tyrosine, and D-alanine. Thereafter, D-aminoacylases were derived from microorganisms belonging to the genus Pseudomonas (Kubo, K., Ishikura, T., and Fukagawa, Y.: J. Antibiot. 43, pp550 (1980), Kubo, K., Ishikura, T. and Fukagawa, Y.: J. Antibiot. 43, pp556 (1980), Kameda, Y., Hase, T., Kanatomo, S. and Kita, Y.: Chem. Pharm. Bull. 26, pp2698 (1978), Kubo, K., Ishikura, T. and Fukagawa, Y.: J. Antibiot. 43, pp543 (1980)), the genus Streptomyces (Sugie, M. and Suzuki, H.: Argric. Biol. Chem. 42, pp107 (1978), Sugie, M. and Suzuki, H.: Argric. Biol. Chem. 44, pp1089 (1980)), and the genus Alcaligenes (Tsai, Y. C., Tseng, C. P., Hsiao, K. M. and Chen, L. Y.: Appl. Environ. Microbiol. 54, pp984 (1988), Yang, Y. B., Hsiao, K. M., Li, H., Yano, Y., Tsugita, A. and Tsai, Y. C.: Biosci. Biotech. Biochem. 56, pp1392 (1992), Yang, Y. B., Lin, C. S., Tseng, C. P., Wang, Y. J. and Tsai, Y. C.: Appl. Environ. Microbiol. 57, pp2767 (1991), Tsai, Y. C., Lin, C. S., Tseng, T. H., Lee, H. and Wang: Microb. Technol. 14, pp384 (1992), Moriguchi, M. and Ideta, K.: Appl. Environ. Microbiol. 54, pp2767 (1988), Sakai, K., Imamura, K., Sonoda, Y., Kido, H. andMoriguchi, M.: FEBS, 289, pp44 (1991), Sakai, K., Obata, T., Ideta, K. and Moriguchi, M.: J. Ferment. Bioeng. 71, pp79 (1991), Sakai, K., Oshima, K. and Moriguchi, M.: Appl. Environ. Microbiol. 57, pp2540 (1991), Moriguchi, M., Sakai, K., Katsuno, Y., Maki, T. andWakayama, M.: Biosci. Biotech. Biochem., 57, pp1145 (1993), Kayama, M., Ashika, T., Miyamoto, Y., Yoshikawa, T., Sonoda, Y., Sakai, K. and Moriguchi, M.: J. Biochem. 118, pp204 (1995)), Moriguchi, M., Sakai, K., Miyamoto, Y. and Wakayama, M.: Biosci. Biotech. Biochem., 57, pp1149 (1993)).
Tsai et al. and Moriguchi et al. also clarified the characteristics of D-aminoacylase derived from microorganisms belonging to the genera Alcaligenes and Pseudomonas and the amino acid and nucleotide sequences of the enzymes. Moriguchi et al. found that microorganisms belonging to the genera Alcaligenes and Pseudomonas produced three species of D-aminoacylase by using different inducers (Wakayama, M., Katsumo, Y., Hayashi, S., Miyamoto, Y., Sakai, K. and Moriguchi, M.: Biosci. Biotech. Biochem. 59, pp2115 (1995)).
Furthermore, Moriguchi et al. determined the nucleotide sequences of these D-aminoacylases derived from a microorganism belonging to the genus Alcaligenes and compared it with L-aminoacylases derived from Bacillus stereothermophilus, human, and pig. The result demonstrated that these D-aminoacylases have a low homology with L-aminoacylases (Wakayama, M., Katsuno, Y., Hayashi, S., Miyamoto, Y., Sakai, K. and Moriguchi, M.: Biosci. Biotech. Biochem., 59, pp2115 (1995)).
Sugie et al. reported D-aminoacylase of a microorganism belonging to the genus Streptomyces of actinomycetes (Sugie, M. and Suzuki, H.: Argric. Biol. Chem. 42, pp107 (1978), Sugie, M. and Suzuki, H.: Argric. Biol. Chem. 44, pp1089 (1980)). However, the enzyme has not been purified yet, and its characteristics remain unknown.
As described above, many D-aminoacylases have been isolated from bacteria and have been used to produce D-amino acids. However, the conventional methods for producing D-amino acids using bacteria have the following problems.
Most of the conventional bacterial D-aminoacylases are inducible enzymes, and N-acetyl-DL-amino acid is usually required for their production. The culture medium of the bacterium used for producing D-aminoacylase contains the unreacted N-acetyl-D-amino acid as well as the degradation product of D-amino acid. In order to react D-aminoacylase produced by these bacteria with a substrate other than N-acetyl-D-amino acid used as the enzyme inducer, it was necessary to isolate the cultured bacteria from the growth medium. Even when the enzyme reacts with N-acetyl-D-amino acid as the substrate, the bacteria must be removed to purify D-amino acid produced. A continuous centrifuge, such as a Westfalia centrifuge, and a Sharples centrifuge were usually used to remove the cultured bacteria. One problem of the centrifugation is that it takes longer to centrifuge a large volume of the culture medium, often causing the inactivation of D-aminoacylase during centrifugation. Furthermore, the bacteria and actinomycetes lyse as the reaction proceeds and thus are difficult to separate centrifugation. As described above, D-amino acids are not always efficiently produced using the conventional bacterial D-aminoacylase.
An objective of the present invention is to provide an efficient method for producing D-amino acids using microorganisms other than the conventional bacteria and actinomycetes.
In order to solve the above-described problems, the present inventors have attempted to produce D-amino acids using fungi which are eukaryotic cells sharing a common cell structure and many chemical properties and functions with animals and plants (eukaryotic characteristics). Fungi are advantageous in that: (1) Fungal cells are large and filamentous and thus can be readily collected by filtration from the liquid culture; (2) fungi efficiently utilize carbohydrates and are able to grow more rapidly than usual at a high carbon/nitrogen ratio, reducing the cost of fermentation materials per unit of enzyme; (3) fungi can be economically cultured in a solid culture (i.e., without a liquid culture) since they grow under natural aeration conditions; this translates into less investment in equipment, high concentration enzyme production, and less organic solvents to extract enzymes; (4) fungi are able to grow well in the solid culture with a low active water level, which can prevent contamination with bacteria that grow poorly in a low active water level; and (5) most fungi efficiently utilize starch, cellulose, and proteins, including inexpensive nutrient sources such as cassava as a starch source, wheat bran, rice, rice bran, barley, wheat, soybean, and cellulose sources.
No fungi producing D-aminoacylase has been reported so far. The present inventors first screened D-aminoacylase-producing fungi and found that many fungi belonging to the genera Hypomyces, Fusarium, Auricularia, Pythium, and Menisporopsis can produce D-amino acid from N-acetyl-D-amino acid. These fungi thus have D-aminoacylase activity.
The present inventors then succeeded in isolating and purifying D-aminoacylases from fungi with D-aminoacylase activity by salting-out with ammonium sulfate and various chromatographies. Furthermore, the inventors studied various physico-chemical properties of the purified D-aminoacylases such as their substrate specificity and thermostability, confirming that D-amino acid can be efficiently produced by reacting the fungal D-aminoacylases with N-acetyl-D-amino acid under the appropriate conditions.
The present inventors are the first to discover D-aminoacylase in eukaryotes, fungi. The enzyme had been found only in prokaryote bacteria and actinomycetes. The fungal D-aminoacylase can be used to produce D-amino acids.
Accordingly, the present invention relates to a D-aminoacylase produced by fungi, a method for producing said D-aminoacylase, and a method for producing D-amino acids using said D-aminoacylase.
More specifically, the present invention provides a D-aminoacylase derived from a fungus.
In another aspect, the invention provides a D-aminoacylase having physico-chemical properties (a) through (f) below:
(a) function: the enzyme acts on N-acetyl-D-amino acids to produce corresponding D-amino acids;
(b) molecular weight: the molecular weight of the enzyme is about 64,000 daltons determined by SDS-polyacrylamide gel electrophores is, and about 56,000 daltons determined by gel filtration chromatography on Superdex 200 Hi-Load 6/16 (Amersham Pharmacia Biotech)
(c) substrate specificity: the enzyme acts on N-acetyl-D-tryptophan, N-acetyl-D-phenylalanine, N-acetyl-D-valine, N-acetyl-D-leucine, and N-acetyl-D-methionine, but not on N-acetyl-L-tryptophan, N-acetyl-L-phenylalanine, N-acetyl-L-valine, N-acetyl-L-leucine, or N-acetyl-L-methionine;
(d) thermostability: when heated at pH 9.5 for 30 min, the enzyme is stable at 45xc2x0 C., but inactivated at 60xc2x0 C. or higher;
(e) optimal temperature: for the reaction at pH 7.5, the enzyme activity is optimal at about 45xc2x0 C.; and
(f) stabilizer: the enzyme activity is stabilized by reducing agents and activated by ICH2CONH2.
The present invention also provides a DNA encoding D-aminoacylase described above.
Furthermore, the invention provides a method for producing D-amino acids, wherein said method comprises reacting a fungus producing D-aminoacylase or D-aminoacylase derived from a fungus with N-acyl-DL-amino acid or its salt represented by the formula (I): 
where R1 and R2 may be identical or different and each represents a hydrogen atom or a substituted or unsubstituted hydrocarbon group, provided that R2 does not represent a hydrogen atom; and X is H, NH4 , or a metal ion.
Moreover, the invention provides a method for producing D-aminoacylase, wherein said method comprises culturing a fungus.